Browsing by Author "Naidoo, Richard."

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Cationic liposome mediated transfection with/without a targeting component.
(2005) Singh, Ashika.; Naidoo, Richard.; Singh, Moganavelli.
The transfer and expression of genes in cells is an important technique for basic research and gene therapy of human disease. A model for gene therapy has been investigated making use of a transfection complex consisting of three components, the DNA i.e. the gene to be transferred and expressed; a gene delivery vehicle viz. a cationic liposome and a cell specific targeting ligand, asialoorosomucoid (AOM). Cationic liposomes are positively charged liposomes that have been prepared from synthetic lipids and have been shown to complex or bind to DNA via electrostatic attraction. They have shown potential as an efficient non-viral gene delivery vehicle in human gene therapy. In this investigation, a novel cationic liposome consisting of 3B [N -(N',N'-dimethylaminopropane)carbamoyl] cholesterol (Chol-T), dioleoylphosphatidylethanolamine (DOPE) and biotinylcholesteryl formylhydrazide was prepared and assessed as a mediator of DNA delivery in a mammalian cell culture system viz. the HepG2 cell line. The cationic liposome was synthesised and characterised by electron microscopy. Foreign DNA may be specifically delivered to target cells by a carrier system which makes use of the recognition of the asialoglycoprotein AOM by cognate receptors on the HepG2 cell plasma membrane. The positively charged AOM was biotinylated and due to this biotinylation, binds streptavidin which contains specific binding sites for biotin. The cationic liposome itself contains biotin residues in its bi-Iayer which in turn binds streptavidin resulting in a ternary complex. Further, due to the DNA binding capability of the cationic liposome, a transfection complex is produced consisting of the three components. The experiments were based on the following concepts: (i) Hepatocytes possess a unique receptor that binds to and internalises galactose-terminal asialoglycoproteins by receptor mediated endocytosis. (ii) Due to electrostatic attraction, DNA binds to cationic liposomes forming soluble complexes. (iii) Through the biotin-streptavidin reaction, the biotinylated AOM is attached to the cationic liposome containing biotin forming complexes enabling targeted delivery of the DNA. (iv) DNA containing the pGL3 gene for the luciferase enzyme was used and following transfection experiments, the luciferase assay was performed to ensure successful transfection. The complexes were tested on the hepatocellular carcinoma cell line, HepG2, which possess the asialoglycoprotein receptor. Transfection studies were conducted using a transient expression system, the luciferase assay system. Some degree of success in the transfection of HepG2 cells was observed. Results obtained in this study suggest that transfection using our targeted transfection complex consisting of cationic liposomes and cell specific targeting ligands does in fact transfect cells by receptor mediation.
Detection of drug metabolizing enzyme gene (DMEs) polymorphisms among the Zulu population of South Africa.
(2007) Makume, Mantha Thandiwe.; Naidoo, Richard.; Naidoo, Kogieleum.; Chelule, Paul Kiprono.
The ability to metabolise drugs and achieve positive therapeutic outcomes is dependent on both genetic and environmental factors. The focus of this study was to determine the distribution and frequency of clinically relevant DME alleles and to assess the impact of these DME alleles on therapeutic outcomes in a cohort of 50 HIV-TB co-infected Zulu participants. PCR-RFLP was used to generate a genotypic profile of CYPIA2, 2C9, 2C19, 2E1, 3A4, MDR-1 and NAT-2. The distributions of the allelic frequencies were as follows. The CYPIA2 (A) - 50.7%, CYP2C9*2 — 100% and *3 — 56.2%, CYP2C19*2 — 35.4%, CYP2E1 (C2) — 28.4%, CYP3A4*1B (G) — 58.2%, MDR-1 (C3435T) - 16% and NAT-2 slow acetylators — 6.5%. Seventy-three percent of participants had prolonged TB therapy. Within this group, 82.9% of patient displayed wild type and 17.2% variant allele for CYP2E1 gene (p= 0.04) profile. In addition, all the slow acetylators in this study had prolonged TB therapy. In the MDR-1 gene, 87.5% showed wild type allele and 12.5% displayed the variant allele. Unsuccessful TB outcomes were also noted in 22% of this study population. In this group the variant allele was found to be dominant in CYPIA2, CYP3A4 and NAT-2, the opposite was seen in CYP2E1 and MDR-1. It was also interesting to note a similar genetic profile in the group that showed successful TB therapy outcomes. All participants had positive ARV treatment outcomes despite DME genotypic variations. However, 26% of all study participants experienced liver enzyme abnormalities. These findings concur with other studies regarding the ethnic distribution of DME alleles and evidence of an association between ART and TB therapeutic outcomes and DME genotype variation was inconclusive.
Epidemiological and genetic risk factors associated with asthma among children in the south Durban region, KwaZulu-Natal.
(2008) Reddy, Poovendhree.; Naidoo, Rajen.; Naidoo, Richard.
Several genes are associated with an increased susceptibility to respiratory diseases, including asthma, which may be exacerbated by ambient air pollution. These genes include the Gluthathione-S-Transferase family (GSTM1 and GST1l) and the NAD(P)H quinone oxidoreductase (NQO1). This, the first genetic epidemiological study conducted in Sub-Saharan Africa had 2 main objectives: I) to evaluate whether the above genotypes confer susceptibility to asthma and related phenotypes; and 2) to investigate if polymorphisms in these genes known to modulate the response to or protect from epithelial oxidative damage modify pulmonary response to ambient air pollutants. A total of 369 schoolchildren from seven primary schools in a heavily industrialized region of south Durban and a demographically similar area in north Durban, Kwa-Zulu Natal, South Africa during the period May 2004 - October 2005, participated in the study. DNA was extracted from whole blood using the GENTRA Puregene kit. Genotyping for the GSTM1 (null vs present genotype) was done using multiplex PCR while the GSTP1 (I1e105Val; AA>AG/GG) and the NQO1(Pro/Ser; CC>CT/TT) genotypes were determined using real time PCR and Taqman probes (Applied Biosystems). Persistent asthma and asthma of "any severity" was determined by questionnaires based on the ATS and BRMC questionnaires. Positive atopy was determined by at least one positive skin test reaction to the seven allergens tested. Other health assessments included spirometry, methacholine challenge testing and four cycles of three-week serial peak flow measurements. Acute respiratory measures included within day variability in FEV1 and PF and the lowest valid values on a given day. SO2. NO2, NO and PM10 were measured over a year using ultraviolet fluorescence, gas-phase chemiluminescence and gravimetric methods respectively. STATA (version 9, College Station, TX, USA) was used for data analysis. Multiple logistic models and Pearson's chi-squared tests were used to evaluate the association between asthma, BHR, atopy and genotype. Covariate-adjusted generalised estimating equations (GEE) with lags of 1-5 days were used to evaluate genotype effect modification of exposure-response. The GSTM1 gene deletion (GSTM1null) was detected in 28.9% of the study population while the distribution of GSTP1 AG/GG and the NQO1 CT/IT polymorphisms were 64.9% and 36.0% respectively. Multiple regression with the adjustment for relevant covariates indicated that individuals carrying one or more copies of the GSTP 1 minor allele had a statistically significant risk for persistent asthma. GSTM1 and NQO1 genotypes showed no significant association with any of the respiratory outcomes tested. However, we found a protective effect for those individuals carrying the GSTM1null genotype and at least one Ser allele (NQO1 CT/TT) for persistent asthma and marked BHR (OR = 0.7, Cl: 0.3-1.5 and OR= 0.3, Cl: 0.0-1.9 respectively). This protective effect is consistent with the role of NQO1 in metabolic activation. Children from the south schools had almost twice the risk of persistent asthma (OR=2.0, Cl: 1.2-3.2, p<.005) and 3 times the risk of BHR (OR=3.5, Cl: 1.4-8.4, p<.005) than those from the schools in the north. Based on symptoms, 20.4% of children from the random sample had persistent asthma and 10.3% had marked BHR (PC20< 2mg/ml). The GEE model results were consistent with modification of air pollutant-pulmonary function relationships by oxidative stress associated genotypes. Statistically significant gene*environment interactions with NO2, NO, and PM10 using FEV1 and PEF outcomes in the expected direction were more frequent for GSTP1 AA and NQO1 CC genotypes (interaction p-values <0.05). There were very few gene*environment interactions for SO2 and any of the 3 SNPs tested. The most striking finding in our study was that pollutant exposure, especially oxides of nitrogen and PM10, even at levels below the recommended limits of South African guidelines, is associated with poorer lung function and that this association is significantly modified by an individual's genotype, particularly the GSTMlnull, GSTPIAA and NQOICC genotypes. Children with the GSTMlnull GSTPI AG/GG, GSTPI AG/GG NQOI CC and GSTMlpos NQOICC gene-gene combinations showed a significant interaction with NO2, NO, and PM10 with decrement in lung function measures. The increased risk to air pollution conferred by the GSTPI and GSTMl genotypes may have clinical and public health importance because this variant is common in most populations. The findings suggest that the risk of developing respiratory symptoms is increased when genetic susceptibility is included with environmental exposures. Our models suggest significant gene*environment interactions i.e the response to the level of air pollutants, as indicated by variability in pulmonary function measures, is modified by genotype. The heightened allergic airway response may be a consequence of a decreased capacity to mount an effective cytoprotective response to oxidative stress. Studying genes may inform us about the biology of asthma which may lead to new therapies or preventative strategies. This study supports the importance of further investigation on these and other genotype variants involved in oxidative stress and respiratory phenotypes in larger cohorts.
A microsatellite evaluation of the genetic status of the p27Kip1 and p21Cip1/WAF1 genes in oesophageal cancer.
(2008) Gaffoor, Zakir.; Naidoo, Richard.
p21 C/P 1/"El and p 2 7K/P 1 are cyclin-dependant kinase inhibitors that fonn an integral part of the cell cycle process. These proteins function as cell-cycle inhibitors, and are able to induce cell cycle arrest by binding to cyclin complexes at key stages. p21 and p27 have been found to be down-regulated in various cancers. This study investigated aberrations at microsatellite markers linked to the p21 and p27 cell cycle genes, in a large cohort of oesophageal squamous cell carcinomas in South Africa. Fluorescent-based PCR were performed on markers linked to both the p21 and p27. The products were run with a 50-500hp marker on 6% denaturing polyacrylamide gels, on the ALFexpresstm' DNA sequencer. The detection and analysis of PCR products was achieved using the AL F e xp res sT M and Fragment M an a aerTm software programmes. Our findings indicate that markers linked to p27 display infrequent aberrations, with loss of heterozygosity ranging from 19% to 37%, and microsatellite instability at 3% to 7%. However, significant relationships between decreased survival time, and aberrations in markers DI2S391 and Dl2S364, were found to exist. Marker D6S1575 linked to p21 displayed frequent allelic loss at 47%, and was comparable to similar studies on the 6p region Further, LOH-Al in this marker was found to be significantly associated with poorly differentiated tumours. The findings from our study indicate that microsatellite aberrations occur infrequently at the p21 and p27 loci in oesophageal cancer. with the exception of marker D6S1575. In addition,this study clearly demonstrates the accuracy and sensitivity of the technology employed. This is the first microsatellite-based investigation of the p21/p27 gene loci in oesophageal cancer in South Africa, using a fluorescent-based PCR assay.
Microsatellite instability in colorectal and oesophageal cancer.
(1998) Naidoo, Richard.; Chetty, Runjun.
The development and progression of carcinogenesis is a major area of interest to many scientists. Numerous factors, including both environmental and genetic have been implicated in the causation of cancer. It is clear that both these factors and others contribute to neoplastic development and progression. Microsatellites are short tandem repeat sequences which are located in the intron segments of the genome. These noncoding sequences range from 2 to 6 base pairs. An increase or decrease in the number of repeat sequences is referred to as microsatellite instability, also referred to as genetic instability. It is thought that microsatellite instability arises as a result of defects in DNA repair process. During DNA synthesis, the DNA repair genes ensure that the correct nucleotide is incorporated into the newly synthesised DNA strand, so when a mismatch base is incorporated, this is promptly removed and replaced with the correct base. However, if the repair system is defective this would give rise to numerous genetic aberrations along that region of the genome. Recently, microsatellite instability and allelic imbalance/loss of heterozygosity have been shown to play an important role in the development of many cancers, especially colorectal cancer (CRC) associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. This study was undertaken to investigate microsatellite instability and allelic imbalance in colorectal and oesophageal carcinomas in the KwaZulu Natal region of South Africa. The molecular analysis was correlated with clinicopathological data to establish a baseline level on which further studies could be performed. In addition, this study represents the first fluorescent based microsatellite analysis of these two common cancers in South Africa. Normal and tumour DNA was isolated from formalin fIxed paraffin embedded tissue. Fluorescent-based DNA technology using an automated DNA sequencer (Alf Express Automated DNA Sequencer) was employed. CY5 labelled primers for microsatellite markers (DCC, D18S34, D18S58, D3S659, D2S123 and D3S1255) were used. The data was captured and analysed using the Fragment Manager Software. The informativity of the microsatellite markers used in this study ranged from 50% to 71.8%. LOH/AI in the region of the DCC gene in the under 35 years of age CRC was 39.1%, while MSI in this region occurred in 31.25% of cases. The DNA repair gene status in these young patients was as follows: LOH/AI: 31.3% and MSI: 40.4%. In the over 50 years of age CRC, LOH/AI in the 18q region was 28% and MSI was 38%. The DNA repair genes (hMSH2 and hMLH1) in this cohort showed LOH/AI in 24% and MSI also in 24%. As regards oesophageal cancer, LOH/AI in the 18q region was 20.5% and MSI 7.7%. The repair genes showed LOH/AI in 17.9% and MSI in 10.25% of cases. When the molecular events were correlated with clinicopathological features, no statistically significant pattern emerged. However, it must be remembered that relatively small numbers of cases (39) were analysed.In conclusion: • No statistical correlation was found between clinicopathological characteristics and the molecular analysis in either CRC and oesophageal cancer. • LOH/AI and MSI was higher in the under 35 age group. • LOH/AI and MSI in 18q, 2p and 3p in sporadic CRC were similar to other fluorescent-based studies in patients over 50 years of age. • LOH/AI and MSI in 18q, 2p and 3p in oesophageal cancer was similar to studies from other geographical areas. • Finally, fluorescent-based microsatellite PCR and analysis was found to be an objective and efficient technique.
Students' understanding of elementary differential calculus concepts in a computer laboratory learning environment at a university of technology.
(2007) Naidoo, Kristie.; Naidoo, Richard.
This thesis investigates the mathematical cognitive errors made in elementary calculus concepts by first-year University of Technology students. A sample of 34 first year students, the experimental group, from the Durban University of Technology Faculty of Engineering were invited to participate in project in elementary calculus using computer technology (CT). A second group, the control group, also consisted of 34 first year engineering students from the same University were given a conventional test in elementary calculus concepts. The experimental group was then given the same conventional test as the control group on completion of the project in elementary calculus using computer technology (CT). The purpose of the analysis was to study the effect of technology on the understanding of key concepts in elementary calculus. The major finding was that technology helps students to make connections, analyse ideas and develop conceptual frameworks for thinking and problem solving. The implications include: • Improvement of curriculum in mathematics at tertiary level; • New strategies for lecturers of elementary calculus; • An improved understanding by students taking the course in elementary calculus. • Redesign of software to improve understanding in elementary calculus.
Studies on the isolation of the polymerase genes from the H1N1 influenza A virus.
(1992) Naidoo, Richard.; Manning, D.
Vaccines directed against the influenza virus become ineffective due to continuous mutation. An alternative approach might be to control replication at the genomic level by enzymatic methylation of the polymerase genes. Hence in this study, a method to locate and successfully isolate the H1N1 influenza A polymerase genes was investigated. The virus was cultured in chick embryos via the allantoic route using aseptic techniques. Following incubation, the allantoic fluid was isolated and washed to remove any contaminating blood cells. The allantoic fluid was checked for fungal and bacterial contamination using the blood agar test and the presence of the virus was established by the haemagglutination titration test. Viral particles were pelleted by ultracentrifugation. Electron microscopy verified the morphology and size of these viruses while immunofluorescence studies, using a monoclonal antibody, confirmed the influenza A strain. The ribose test verified the presence of RNA in the samples. Purified viral pellets were pooled and homogenised in buffer containing guanidine thiocyanate, mercaptoethanol and sarkosyl. The samples were incubated on ice before mechanical disruption of the virus. Viral RNA was isolated from the upper aqueous layer after a standard phenol/chloroform extraction procedure. RNA was quantified spectrophotometrically and purity assessed initially by the absorbance ratio readings at 260/280 nm. Electrophoresis of the RNA samples was performed together with RNA molecular weight markers on a 1.5% formamide agarose gel. Five bands were identified and the band containing the polymerase genes was size selected, located and excised. Purification of the polymerase genes from the agarose was achieved by using the BIO 101 RNAid kit. The three isolated polymerase RNAs were reverse transcribed using the Boehringer Mannheim cDNA synthesis kit. The results indicate that the H1N1 influenza virus was successfully grown and isolated from chick embryos. Absence of contamination and verification of viral presence at different stages of the study were indications that asepsis was successfully achieved. The RNA obtained was sufficient and suitable for cDNA synthesis. This cDNA may now be used for further molecular analysis and subsequent DNA methylation studies. Further, transfection studies may then be performed to determine, if any, the the expression of methylated and unmethylated cDNA.